Journal: Oncology Letters

Article Title: SPAG6 silencing induces autophagic cell death in SKM-1 cells via the AMPK/mTOR/ULK1 signaling pathway

doi: 10.3892/ol.2020.11607

Figure Lengend Snippet: Inhibiting autophagy with autophagy inhibitors attenuates SPAG6 knockdown-induced apoptosis. Cells were treated with SPAG6-shRNA lentivirus or NC-shRNA lentivirus alone, or co-treated with 3-MA or CQ and SPAG6-shRNA. (A) Relative protein expression levels of SPAG6, LC3, Beclin1 and P62 in each group were detected via western blot analysis. (B) Transmission electron microscopy revealed the intracellular ultrastructures of each group (magnification, ×20,000). The images display a representative experiment from three independent experiments. Red arrows indicate autophagic vacuoles. Scale bar, 1 µm. (C) Number of autophagosomes per field was quantified. (D) Relative protein expression levels of SPAG6, Bcl-2, Bax and cleaved caspase-3 in each group were detected using western blot analysis. (E) Following annexin V APC/DAPI double-staining, the total apoptosis rate of SKM-1 cells treated with NC-shRNA or SPAG6-shRNA alone and SKM-1 cells co-treated with SPAG6-shRNA and 3-MA or CQ was detected using flow cytometry. The images display a representative experiment from three independent experiments. (F) Analysis of the apoptotic rate shown in (E) Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 vs. NC-shRNA, # P<0.05; ## P<0.01 vs. SPAG6-shRNA. NC, negative control; shRNA, short hairpin RNA; SPAG6, sperm-associated antigen 6; LC3, microtubule-associated protein 1 light chain 3; Beclin1, autophagy-associated protein 6; p62, sequestosome 1; CQ, chloroquine; 3-MA, 3-methyladenine; APC, allophycocyanin; LL, lower left; UL, upper left; LR, lower right; UR, upper right; ns, not significant.

Article Snippet: Cells were grown in complete medium with 3-methyladenine (3-MA; 5 mM/l), chloroquine (CQ; 10 μM/l) or the AMPK inhibitor Compound C (5 μM/l), purchased from Selleck Chemicals and all cells were incubated at 37°C with 5% CO 2 .

Techniques: Knockdown, shRNA, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Double Staining, Flow Cytometry, Standard Deviation, Negative Control

Journal:

Article Title: Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis

doi: 10.1073/pnas.022641099

Figure Lengend Snippet: Modulation of CIA in the rat by prednisolone and the GC derivatives. (A) Chemical structure of prednisolone and the chains added to position 21 to yield either NCX-1015 or NCX-1016. (B–D) Rats were treated daily i.p. with NCX-1015 (▴, 4 μmol/kg; ▵, 0.4 μmol/kg), prednisolone (●, 4 μmol/kg; ○, 0.4 μmol/kg), NCX-1016 (♦, 4 μmol/kg) or vehicle (■, 100 μl peanut oil) from day 12 to day 18 after collagen II injection. A group of naïve rats (□) also was used. During the development of the arthritis, plethysmometry was used to assess paw volume (B), and rats' paws and ankles were individually assessed and scored (C), as described in Materials and Methods. After killing the animals (day 18), anklebone thickness was measured with calipers (D). Data are expressed as means ± SEM (n = 10 per group). *, P ≤ 0.05 vs. vehicle-treated group and #, P ≤ 0.05 vs. naïve animals.

Article Snippet: The negative control compound, deprived of the nitrooxy group, NCX-1016 (Mw 494.6) was synthesized at NicOx laboratories in Milan, Italy, as follows: prednisolone (33.3 mmol in tetrahydrofurane) was added to 49.9 mmol of 4-(hydroxymethyl)benzoyl chloride and triethylamine and stirred for 24 h at room temperature.

Techniques: Injection

Journal:

Article Title: Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis

doi: 10.1073/pnas.022641099

Figure Lengend Snippet: Effect of CIA and GC treatment on serum levels of cytokines and pyridinoline. (A–C) Prednisolone (Pred), NCX-1015, NCX-1016, or vehicle (V) treatment was the same as in Fig. ​Fig.11 (i.p. from day 12 at the indicated doses in μmol/kg), whereas in D, drugs or vehicle (V) were given orally. A group of naïve rats also was used. (A) IL-1β, (B) IL-6 and (C and D) pyridinoline concentration as measured by using specific ELISA. Data are expressed as means ± SEM (n = 10 per group). *, P ≤ 0.05 vs. vehicle-treated group and #, P ≤ 0.05 vs. naïve animals.

Article Snippet: The negative control compound, deprived of the nitrooxy group, NCX-1016 (Mw 494.6) was synthesized at NicOx laboratories in Milan, Italy, as follows: prednisolone (33.3 mmol in tetrahydrofurane) was added to 49.9 mmol of 4-(hydroxymethyl)benzoyl chloride and triethylamine and stirred for 24 h at room temperature.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis

doi: 10.1073/pnas.022641099

Figure Lengend Snippet: Bone resorption measured in rat primary osteoclasts. A mixed population of rat bone cells was incubated overnight with prednisolone (Pred), NCX-1015, NCX-1016, or vehicle (V). Osteoclast activity was measured as described in Materials and Methods. (A) Osteoclast bone resorptive activity after 18-h incubation with increasing concentrations of prednisolone. *, P < 0.05 vs. vehicle (dose 0). (B) NCX-1016 mimicked prednisolone effect, whereas NCX-1015 was inactive. *, P < 0.05 vs. vehicle-treated cells. (C) Addition of the NO-donor NOC-18 (1 μM) to 1 nM prednisolone or NCX-1016 abolished the increase in bone resorption. (D) In the presence of the guanylate cyclase inhibitor (ODQ, 5 μM), 1 nM NCX-1015 stimulated osteoclast activity. All results are the means ± SEM of at least three to four experiments performed in triplicate.

Article Snippet: The negative control compound, deprived of the nitrooxy group, NCX-1016 (Mw 494.6) was synthesized at NicOx laboratories in Milan, Italy, as follows: prednisolone (33.3 mmol in tetrahydrofurane) was added to 49.9 mmol of 4-(hydroxymethyl)benzoyl chloride and triethylamine and stirred for 24 h at room temperature.

Techniques: Incubation, Activity Assay

Journal: PLoS ONE

Article Title: Characterization of a Novel Small Molecule Subtype Specific Estrogen-Related Receptor α Antagonist in MCF-7 Breast Cancer Cells

doi: 10.1371/journal.pone.0005624

Figure Lengend Snippet: Short hairpin plasmids expressing short hairpin RNA (shRNA) specific for knocking-down ERRα were transfected separately into MCF-7 cells. In addition, a shRNA expressing a scrambled artificial non-specific sequence was transfected. (-) control. (A) MCF-7/shRNA ERRα3 cells underwent four rounds (I–IV) of fluorescent activated cell sorting (FACS) to enrich for GFP expressing cells. FACS is described in Materials and Methods . (B) ERRα ( ESRRA ), ACADM , and PGC-1α ( PPARGC1A ) mRNA expression was measured by real-time RT-PCR. After 4 rounds of FACS, MCF-7/shRNA ERRα3 cells ERRα ( ESRRA ) mRNA levels were significantly reduced by 79%, ACADM levels by 75%, and PGC-1α ( PPARGC1A ) by 71% (*, P<0.05). Differences in relative mRNA expression between MCF-7/shRNA (-) and MCF-7/shRNA ERRα3 were measured by ANOVA followed by a student t-test with a 0.05 significance level. (C) MCF-7, MCF-7/shRNA (-), MCF-7/shRNA ERRα2, and MCF-7/shRNA ERRα3 cells ERRα protein expression was measured by Western blot, equal loading of protein was assessed by Coomassie blue staining of gels, and densitometric quantification are described in Materials and Methods . Results shown are representative of three independent experiments. MCF-7/shRNA ERRα2 exhibited 59% less protein verses the negative control, while MCF-7/shRNA ERRα3 cells ERRα protein levels were reduced by 69%. (D) ERα ( ESR1 ), aromatase ( CYP19A1 ), osteopontin ( SPP1 ), and pS2 ( TFF1 ) mRNA expression was measured in MCF-7/shRNA (-), MCF-7/shRNA ERRα2, and MCF-7/shRNA ERRα3 cells by real-time RT-PCR after 4 rounds of FACS. Knocking-down ERRα by shRNA (RNAi) led to significant decrease (*, P<0.05) in expression of ERRα target genes aromatase ( CYP19A1 ), osteopontin ( SPP1 ), and pS2 ( TFF1 ) while ERα ( ESR1 ) levels were not affected. All real-time RT-PCR results are representative of three independent experiments performed in triplicate.

Article Snippet: Primer/probe sets for target genes: human ERRα ( ESRRA ) (Hs00607062_gH), human ERα ( ESR1 ) (Hs00174860_m1), human PGC-1α ( PPARGC1A ) (Hs00173304_m1), human PDK4 ( PDK4 ) (Hs00176875_m1), human osteopontin ( SPP1 ) (Hs00959010_m1), human pS2 ( TFF1 ) (Hs00170216_m1), human ACADM ( ACADM ) (Hs00163494_m1) and 18S rRNA endogenous control (4308329) were purchased from Applied Biosystems.

Techniques: Expressing, shRNA, Transfection, Sequencing, Control, FACS, Quantitative RT-PCR, Western Blot, Staining, Negative Control

Journal: Radiation research

Article Title: UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells.

doi: 10.1667/rr2445.1

Figure Lengend Snippet: FIG. 2. Panel A: Expression level of EGFR protein in Panc1, KP3 and PE cells. The cells were harvested and protein extracts (5 mg each) were analyzed by Western blotting. The upper and middle panels present the results after short and long exposure to film, respectively. Panel B: UVC radiation caused a decrease in EGFR protein levels in Panc1, KP3 and PE cells. The cells were exposed to UVC radiation at 200 J and then incubated for the indicated periods. Protein extracts were then harvested and examined by Western blotting using anti- EGFR and anti-GAPDH antibodies. Panel C: Quantification of the Western blotting results shown in Fig. panel A. The asterisks (*, **) indicate statistically significant difference (P , 0.05) compared to PE cells.

Article Snippet: In brief, Panc1 and KP3 cells were pretreated with the indicated compounds and then exposed to the mouse anti-EGFR antibody (Santa Cruz Biotechnology) that recognizes the extracellular domain of the EGFR (1:50 dilution) in DMEM containing 1% bovine serum albumin (BSA) for 15 min at 37uC.

Techniques: Expressing, Western Blot, Incubation

Journal: Radiation research

Article Title: UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells.

doi: 10.1667/rr2445.1

Figure Lengend Snippet: FIG. 3. UVC radiation caused the phosphorylation of EGFR at S1046/7 in pancreatic cancer cells. Panc1 (panel A) and KP3 (panel B) cells were exposed to UVC radiation at 200 J and then incubated for the indicated periods. Protein extracts were then harvested and examined by Western blotting using anti-phospho-EGFR at S1046/7, Y992, Y1045, Y1068 and Y1173, anti-EGFR and anti-GAPDH antibodies. The each lower bar graph shows quantification data for the phosphorylation levels of EGFR after normalization with respect to GAPDH.

Article Snippet: In brief, Panc1 and KP3 cells were pretreated with the indicated compounds and then exposed to the mouse anti-EGFR antibody (Santa Cruz Biotechnology) that recognizes the extracellular domain of the EGFR (1:50 dilution) in DMEM containing 1% bovine serum albumin (BSA) for 15 min at 37uC.

Techniques: Phospho-proteomics, Incubation, Western Blot

Journal: Radiation research

Article Title: UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells.

doi: 10.1667/rr2445.1

Figure Lengend Snippet: FIG. 5. The inhibition of p38 MAPK suppressed UVC-radiation-induced phosphorylation of EGFR at S1046/7. Panc1 (panel A) and KP3 (panel B) cells were pretreated with 20 mM of SB203580 or 1 mM of BIRB0790 for 1 h, exposed to UVC radiation at 200 J, and then incubated for 1 h. Protein extracts were then harvested and examined by Western blotting using anti-phospho-EGFR at S1046/7, anti-EGFR and anti- GAPDH antibodies. The lower bar graph shows quantification data for the relative phosphorylation levels of EGFR at S1046/7 after normalization with respect to GAPDH. Panels C and D: Effect of gene silencing with p38 MAPK-siRNA transfection on pancreatic cancer cells. Panc1 (panel C) and KP3 (panel D) cells were incubated with 100 nM of p38 MAPK-siRNA or negative control (NC)-siRNA, followed by incubation for 1 h after exposure to UVC radiation (200 J). Protein extracts were then prepared and examined by Western blotting using anti-phospho-EGFR at S1046/7, EGFR, p38 MAPK, p44/p42 MAPK, SAPK/JNK and Akt. An antibody to GAPDH was used as the control for protein loading. The lower bar graph shows quantification data for the relative phosphorylation levels of EGFR at S1046/7 after normalization with respect to GAPDH. Representative results from triplicate independent experiments with similar results are shown. NC indicates negative control.

Article Snippet: In brief, Panc1 and KP3 cells were pretreated with the indicated compounds and then exposed to the mouse anti-EGFR antibody (Santa Cruz Biotechnology) that recognizes the extracellular domain of the EGFR (1:50 dilution) in DMEM containing 1% bovine serum albumin (BSA) for 15 min at 37uC.

Techniques: Inhibition, Phospho-proteomics, Incubation, Western Blot, Transfection, Negative Control, Control

Journal: Radiation research

Article Title: UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells.

doi: 10.1667/rr2445.1

Figure Lengend Snippet: FIG. 6. EGFR internalization and phosphorylation at S1046/7 induced by UVC radiation were canceled by the treatment with SB203580 in Panc1 (panel A) and KP3 (panel B) pancreatic cancer cells. The cells were pretreated with or without 20 mM SB203580 for 1 h and then labeled with anti-EGFR antibodies. Thirty minutes after exposure to UVC radiation (200 J), they were fixed, permeabilized and treated with anti-phospho-EGFR at S1046/7 antibodies followed by Alexa 546-conjugated secondary antibodies for EGFR (red signal) and Alexa 488 conjugated secondary antibodies for phospho-EGFR at S1046/7 (green signal) and DAPI (blue signal) and then examined by fluorescence microscopy.

Article Snippet: In brief, Panc1 and KP3 cells were pretreated with the indicated compounds and then exposed to the mouse anti-EGFR antibody (Santa Cruz Biotechnology) that recognizes the extracellular domain of the EGFR (1:50 dilution) in DMEM containing 1% bovine serum albumin (BSA) for 15 min at 37uC.

Techniques: Phospho-proteomics, Labeling, Fluorescence, Microscopy

Journal: Radiation research

Article Title: UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells.

doi: 10.1667/rr2445.1

Figure Lengend Snippet: FIG. 7. Panel A: UVC radiation induced a decrease in the cell surface amount of EGFR. Line graph shows quantification data for cell surface amounts of EGFR analyzed by ELISA (see the Materials and Methods). #, Panc1 without UVC irradiation; D, KP3 without UVC irradiation;N, Panc1 cells exposed to 30 J of UVC radiation; m, KP3 cells exposed to 30 J of UVC radiation. Panel B: EGFR internalization induced by UVC radiation was blocked by treatment with concanavalin A (Con A), an endocytosis inhibitor, in Panc1 cells. The Panc1 cells were pretreated with or without 100 mg/ml of Con A for 1 h and then labeled with anti-EGFR antibodies. After exposure to UVC radiation for 30 min, they were fixed and permeabilized, treated with Alexa 488-conjugated secondary antibod- ies for EGFR (green signal) and DAPI (blue signal), and then examined by fluorescence microscopy. The asterisks (*, **) indicate statistically significant differences (P , 0.05) compared to PE cells.

Article Snippet: In brief, Panc1 and KP3 cells were pretreated with the indicated compounds and then exposed to the mouse anti-EGFR antibody (Santa Cruz Biotechnology) that recognizes the extracellular domain of the EGFR (1:50 dilution) in DMEM containing 1% bovine serum albumin (BSA) for 15 min at 37uC.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Labeling, Fluorescence, Microscopy

Journal: Radiation research

Article Title: UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells.

doi: 10.1667/rr2445.1

Figure Lengend Snippet: FIG. 8. Panel A: The internalized EGFR induced by UVC radiation is not associated with c-Cbl. Panc1 cells were first labeled with an anti-EGFR antibodies as described above and then treated with EGF (30 ng/ml) or UVC radiation (200 J), followed by fixation and permeabilization. The cells were then exposed to anti-c-Cbl antibodies followed by Alexa 546-conjugated secondary antibodies for EGFR (red signal), Alexa 488-conjugated secondary antibodies for c-Cbl (green signal), and DAPI (blue signal) and then examined by fluorescence microscopy. Panel B: The internalized EGFR was colocalized with EEA-1. Panc1 cells were first labeled with anti- EGFR antibodies then treated with EGF (30 ng/ml) or UVC radiation (200 J), followed by fixation and permeabilization. The cells were then exposed to anti-EEA-1 antibodies, followed by Alexa 546 conjugated secondary antibodies for EGFR (red signal), Alexa 488-conjugated secondary antibodies for EEA-1 (green signal), and DAPI (blue signal) and then examined by fluorescence microscopy. Representative results from at least three independent experiments are shown.

Article Snippet: In brief, Panc1 and KP3 cells were pretreated with the indicated compounds and then exposed to the mouse anti-EGFR antibody (Santa Cruz Biotechnology) that recognizes the extracellular domain of the EGFR (1:50 dilution) in DMEM containing 1% bovine serum albumin (BSA) for 15 min at 37uC.

Techniques: Labeling, Fluorescence, Microscopy